Transcriptional Regulatory Mechanisms And Effects Of Indole On Poly(A) Polymerase I In Pantoea Agglomerans YS19

Confronted with fluctuantenvironments,bacteria have evolved numerous regulating mechanisms,of which indole-based one is drawing wide attention.Indole has been found to be engaged in regulating multiple physiological characteristics including bacterial motility,stress resistance,and colonizationin plant,but the mechanism behind remains unknown.Pantoea agglomerans YS19 was a predominant endophyte isolated from rice.It was charactered to form a special aggregate structure called symplasmata.We previously screened a indole-associated?pcnB mutant by m Tn5 transponson mutagenesis approach and found that the mutant gene pcnB,encoding the Poly(A)polymerases(PAPI),a global regulatory factor,played a crucial role indole-based regulation in YS19.This provides a good entrance for the regulatory mechanism study of indole in YS19.Therefore,revealing of the regulatory mechanism of indole on pcnB transcription is of great significance.This study detected the regulatory effects of indole on the stress resistance of YS19and the?pcnB mutantand illustrated that the mutation of pcnBhas a significant influence on the stress resistance of YS19 and also on its response to indole.In the mutant,the resistance to starvation was inhibited(decreased by 45.20%at 8 d),whereas the resistance to tetracycline(increased by 387.69%)and ultraviolet radiation(increased by 200.33%)was increased.In the anti-tetracycline experiment,the survival of wild-type and mutant group was increased by 113.02%and 59.67%respectively when indole added;in the UV-resistant experiment,the survival of wild-type group was increased by 123.99%as indole added,but had no significant effect on the mutant.In the experiment of anti-hunger,adding indole increased the survival of wild-type group by 19.01%(8 d),but had no effect on the mutant.The final concentration of indole added in this three experiments was 0.5 m M.These data suggested the complexity of the indole regulation.In the regulation,pcnB gene did play a key role,but there might also contain other pathways in the indole regulation as a compensation of the mutation of pcnB.We then constructed a series of recombinant plasmids containing the promoter of pcnB gene by molecular biological technique.gfpwas used as the reporter gene to investigate the regulatory effects of indole on the pcnB promoter.In E.coli for the work,indole enhanced the activity of pcnB promoter.By fragment deletion experiments,it was confirmed that the core region of the promoter of pcnB gene in YS19 is the?⁷⁰-dependent promoter p B;the core sequence(In-pcnB)in response to indole regulation is located in the upstream of TIS-127 bp to-88 bp,approximately 40 bp.In addition,recombinant plasmids were transformed into Pantoea ananatis YJ76,a near evolutionary relation strain of YS19,and showed that indole inhibited the activity of pcnB promoter,indicating astrain-specificity of the pcnB promoter in indoleresponse.Furthermore,the promoter region of a gene zurthat encoding Zur protein as a zinc-dependent transcription inhibitor in E.coli was verified to have homologus fragment(%)to In-pcnB core region.In E.coli,indole was tested to have the same regulatory effect on the promoter of this zur gene as the pcnB promoter.However,it was not responsive in YJ76,further confirming the strain-specificity of indole regulation in bacteria.This study explored the regulatory mechanism of indole on PAPIin transcription levels and preliminarily obtained the regulatory pattern of indole towards the promoter region of pcnB.Not only did it lead to new understanding of the mechanism on bacterial stress resistance and but also the regulation mechanismof indole,which enriched our knowledge on the polyadenylation regulation in bacteria and its biological functions.